HNO plays significant roles in many biological processes, such as vascular relaxation, enzyme activity regulation, and neurological function regulation. It offers a promising new treatment for diseases such as heart failure and stroke. The widespread biomedical effects of HNO have promoted the idea that it is a signaling molecule. However, the potential in vivo formation pathways still need to be identified and validated. Given plentiful reports of conversion between HNO and NO via metalloproteins, most working mechanisms are yet to be disclosed, to help investigate relationships between these two important molecules and help design good HNO scavengers. Development of direct, fast, and selective in vivo HNO fluorescence probes to detect its real-time biological actions are quite challenging, with most mechanistic information still unknown. The long-term goal of our research is to provide accurate mechanistic information of biological HNO formation, conversion, and detection via metalloproteins and models. The first objective is to provide mechanistic profiles of the experimentally found heme protein mediated HNO formation from hydroxylamines, intermediates from nitric oxide synthase catalytic turnover. Both quantum mechanics (QM) and hybrid QM and molecular mechanics (QM/MM) methods will be used to have a systematic study of different heme proteins and different substrates for biological HNO formation. Results will help evaluate potential in vivo precursors for HNO, and facilitate studies of the pharmaceutical effects of some HNO donors to treat various diseases. The second objective is to use both QM and QM/MM methods to understand the effects from different metal environments, active site residues, and protein environments on HNO/NO conversion via non-heme proteins, an important basis for relevant in vivo formation of NO and regulation of HNO/NO concentrations. Results will also assist the use of the mechanistic information of different metal centers and ligands to develop rational guidelines to design fast HNO detection/trapping agents. The third objective is to provide unprecedented mechanistic profiles to understand the experimental reactivity and selectivity results of some metal-based HNO fluorescence probes with different structural motifs and selectivity patterns. A systematic QM study of these systems and their involved reactions will be performed to obtain detailed information to understand experimental high reactivity and selectivity origins and develop rational guidelines to facilitate future development of highly selective and fast HNO probes. Overall, these studies will help understand the significant roles of HNO in cellular signaling and regulation and related therapeutic treatments.